Microglia are tissue macrophages that reside in the central nervous system (CNS) and perform unique and critical auxiliary functions important to CNS development, homeostasis, immunity and repair. Despite their importance, remarkably little is known about the mechanisms that control microglia development and function, particularly in humans. Here we propose to leverage recent technical and computational breakthroughs to investigate how interactions between lineage-determining and signal-dependent transcription factors regulate gene expression in human and mouse microglia. Specific Aim 1 will test the hypothesis that the human brain microenvironment is an important determinant of microglia gene expression. Microglia transcriptomes will be obtained immediately upon isolation and after one week in a tissue culture environment supplemented with putative regulators of microglia phenotypes. Importantly, all samples will be analyzed in the context of full genomic sequence. These studies will provide qualitatively new information on core transcriptional signatures of microglia, the influence of the brain microenvironment, and the relationships of genotype to gene expression patterns. Specific Aim 2 will test the hypothesis that PU.1 and a limited set of alternative lineage determining factors drive the selection of the majority of enhancers that determine microglia identity and regulatory potential. We will use ChIP-Seq and ATAC-Seq methods to define enhancer atlases in freshly isolated human microglia. This information will suggest key microglia lineage determining factors and identify microglia super enhancers. We will exploit the combination of ChIP sequencing and natural genetic variation to identify core transcription factor combinations needed for the selection and activation of microglia-specific enhancers. These findings will provide qualitatively new insights into molecular mechanisms that underlie human microglia development and function and will inform efforts to more faithfully reprogram human iPS cells to microglia phenotypes. Specific Aim 3 will test the hypothesis that environmental factors and transcriptional circuits required for microglia-specific gene expression are conserved between mouse and human. Mouse microglia transcriptomes and enhancer atlases will be obtained under the same conditions as described for human microglia in Specific Aims 1 and 2. Different strains of mice will be used as a source of natural genetic variation to define core transcription factor combinations needed for enhancer selection and activation. Roles of specific transcription factors predicted to have conserved roles in regulating human and mouse microglia development and function will be assessed by loss of function experiments. These experiments will provide important information on the extent to which mouse microglia can be used to model human microglia and establish the functional importance of conserved transcription factors.